INTRODUCTION CD44 is a widely expressed cell surface glycoprotein that has been implicated in a variety of cellular functions involving cell adhesion and migration, and in the metastasis of certain cancers (reviewed in Lesley et al., 1993a). Hyaluronan (HA) is a high molecular mass glycosaminoglycan (GAG), which has been identified as a major ligand for CD44 (Aruffo et al., 1990; Culty et al., 1990; Lesley et al., 1990; Miyake et al., 1990b). The interaction between HA and CD44 promotes cell-cell and cell-matrix interactions and CD44 has been implicated in bone marrow-stromal cell interactions (Miyake et al., 1990a,b), lymphocyte-endothelial cell interactions (Aruffo et al., 1990; Lesley et al., 1992; Camp et al., 1993; Zahalka et al., 1995; Clark et al., 1996; DeGrendele et al., 1996), angiogenesis (Trochon et al., 1996) and in arterial wall repair (Jain et al., 1996). As with other cell surface molecules involved in cell adhesion and cell migration, the adhesive property of CD44 is tightly regulated. Three different binding states have been identified for CD44 binding to HA: constitutive, inducible and non-binding (Lesley and Hyman, 1992) . In general, cells expressing CD44 do not constitutively bind HA; however, some cells can be induced to bind HA after an in vivo allogenic reaction (Lesley et al., 1994), an in vivo chronic graft-versus- host reaction (Murakami et al., 1991), an in vitro induction by interleukin-5 in B cells (Murakami et al., 1990) and after arterial wall injury (Jain et al., 1996). Phorbol myristate acetate (PMA), which can mimic aspects of T cell activation, increases the expression of CD44 and induces the binding of CD44 to HA (Lesley et al., 1990; Hyman et al., 1991; Liao et al., 1993). The inducible binding state of CD44 can be identified using the anti-mouse CD44 specific monoclonal antibody (mAb), IRAWB 14, which can bind CD44 and induce HA binding (Lesley et al., 1992). CD44 can exist in multiple isoforms generated by alternative splicing of the membrane proximal region of the molecule, which provides additional sites for glycosylation and GAG addition (Screaton et al., 1992). The standard form of CD44, CD44H, contains none of the alternatively spliced exons and exhibits different HA binding abilities, depending upon the cell type or the activation state of the cell (Lesley et al., 1990; and reviewed in Lesley et al., 1993a). Within a particular cell line or cell type, the alternatively spliced forms of CD44 can exhibit different HA binding abilities from the standard, CD44H form (Stamenkovic et al., 1991; Liao et al., 1993; Bennett et al., 1995; Jackson et al., 1995; van der Voort et al., 1995; Sleeman et al., 1996). Both N- and O-linked glycosylations have also been shown to influence the HA binding ability of CD44 in both a cell type- and CD44 isoform-specific manner. The inhibition of N-linked glycosylation in a variety of cell lines 1021 Journal of Cell Science 111, 1021-1029 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS7129 CD44 is a widely expressed cell adhesion molecule that binds the extracellular matrix component, hyaluronan, in a tightly regulated manner. Previous studies have shown that the CD44-hyaluronan interaction is affected by changes in the glycosylation state of CD44. In this study, we take advantage of several well-characterized murine L cell mutants defective in heparan sulfate synthesis (gro2C cells), heparan sulfate and chondroitin sulfate synthesis (sog9 cells), and glycosaminoglycan and oligosaccharide processing (sog8 cells) to assess the effects of these defects on the hyaluronan binding ability of CD44. In parental L cells and gro2C cells, CD44 was induced to bind hyaluronan after addition of the activating, anti-CD44 monoclonal antibody, IRAWB 14. By contrast, no inducible binding was observed in sog9 cells. Treatment of L cells with sodium chlorate, an inhibitor of sulfation, also abolished inducible hyaluronan binding. However, inducible and some constitutive hyaluronan binding was observed in sog8 cells. This indicates that sulfation and, in particular, the addition of chondroitin sulfate are required for inducible hyaluronan binding by CD44 in L cells. However, in the absence of fully processed oligosaccharides, chondroitin sulfate is not essential for hyaluronan binding, indicating that the effect of chondroitin sulfate is dependent upon the glycosylation state of the cell. Thus, in addition to glycosylation, chondroitin sulfate biosynthesis is an important post-translational modification that can affect the hyaluronan binding ability of CD44. Key words: CD44, Hyaluronan, Chondroitin sulfate, Glycosylation SUMMARY Analysis of CD44 interactions with hyaluronan in murine L cell fibroblasts deficient in glycosaminoglycan synthesis: a role for chondroitin sulfate Lesley E. Esford, Arpita Maiti, Sharon A. Bader, Frank Tufaro and Pauline Johnson* Department of Microbiology and Immunology, University of British Columbia, Vancouver, B.C. V6T 1Z3 Canada *Author for correspondence (E-mail: Pauline@unixg.ubc.ca) Accepted 16 January; published on WWW 9 March 1998